Structure and Function of the Universal Stress Protein TeaD and Its

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Biochemistry 2010, 49, 2194–2204 DOI: 10.1021/bi9017522

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Structure and Function of the Universal Stress Protein TeaD and Its Role in Regulating the Ectoine Transporter TeaABC of Halomonas elongata DSM 2581T† Eva S. Schweikhard,‡,^ Sonja I. Kuhlmann,‡,^ Hans-J€org Kunte,§, Katrin Grammann, and Christine M. Ziegler*,‡ ‡

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Department of Structural Biology, Max Planck Institute of Biophysics, Max-von-Laue-Strasse 3, 60438 Frankfurt am Main, Germany, §Biology in Materials Protection and Environmental Issues, Federal Institute for Materials Research and Testing (BAM), Unter den Eichen 87, 12205 Berlin, Germany, and Institute for Microbiology and Biotechnology, University of Bonn, Meckenheimer Allee 168, 53115 Bonn, Germany^Both authors have equally contributed to the work Received October 11, 2009; Revised Manuscript Received January 10, 2010 ABSTRACT:

The halophilic bacterium Halomonas elongata takes up the compatible solute ectoine via the osmoregulated TRAP transporter TeaABC. A fourth orf (teaD) is located adjacent to the teaABC locus that encodes a putative universal stress protein (USP). By RT-PCR experiments we proved a cotranscription of teaD along with teaABC. Deletion of teaD resulted in an enhanced uptake for ectoine by the transporter TeaABC and hence a negative activity regulation of TeaABC by TeaD. A transcriptional regulation via DNA binding could be excluded. ATP binding to native TeaD was shown by HPLC, and the crystal structure of TeaD was solved in complex with ATP to a resolution of 1.9 A˚ by molecular replacement. TeaD forms a dimer-dimer complex with one ATP molecule bound to each monomer, which has a Rossmann-like R/β overall fold. Our results reveal an ATP-dependent oligomerization of TeaD, which might have a functional role in the regulatory mechanism of TeaD. USP-encoding orfs, which are located adjacent to genes encoding for TeaABC homologues, could be identified in several other organisms, and their physiological role in balancing the internal cellular ectoine pool is discussed.

The halophilic proteobacterium Halomonas elongata can tolerate salt concentrations well above 100 g/L (1.72 M) NaCl (1). To avoid dehydration in such environments and to maintain an osmotic equilibrium, H. elongata amasses internally large quantities of compatible solutes. These highly water-soluble molecules do not disturb the cell metabolism even at molar concentrations and are used for osmoregulation by diverse groups of bacteria originating from various environments (2). A clear trend was observed in which the least salt-tolerant organisms synthesize disaccharides for osmoregulation, whereas marine (halotolerant) species accumulate sugar polyols, and halophilic bacteria employ nitrogen containing compatible solutes such as glycine betaine and ectoine (3, 4). H. elongata synthesizes ectoine as its main compatible solute (5) but does not rely only on de novo synthesis. It can also take up ectoine from the medium. H. elongata is equipped with only one ectoine-specific transporter TeaABC (6), which was shown to be a new type of osmoregulated transport system belonging to the family of tripartite ATP-independent periplasmic transporter (TRAP-T) (6) evolutionarily placed between the families of secondary and ABC transporters. The key distinguishing feature of TRAP transporters is the presence of a periplasmic substrate binding protein (SBP) as found in ABC systems, but with transport being driven by an electrochemical ion gradient as found in secondary transporters. TRAP-Ts have two integral membrane subunits, a 12 TM helix subunit (TeaC) that probably harbors the substrate and ion binding sites and a 4 TM helix protein (TeaB) of unknown but essential function. These subunits can also be found as a single fused protein (7). † The work was funded by DFG KU 1112/3-1 and ZI 572/4-1. *To whom correspondence should be addressed. Telephone: þ49 (69) 6303-3054. Fax: þ49 (69) 6303-3002. E-mail: christine.ziegler@ mpibp-frankfurt.mpg.de.

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Published on Web 01/29/2010

The physiological role of TeaABC is not only to import exogenous ectoine from the environment but also to salvage endogenous ectoine leaking through the cytoplasmic membrane, which would be otherwise lost to the medium (6). A 15.5 kDa universal stress protein (USP)1 encoding orf of 441 bp was found immediately downstream of teaC (6) and was named teaD accordingly. Genes encoding homologues of USPs adjacent to a transporter have been observed not only in TRAP systems (7) but also for a wide range of secondary transporter and channel genes (8), but their role in transport has not yet been explored (8). This might also be related to the fact that although USPs form an ancient and conserved family of proteins found in all kingdoms of life (9), their biochemical activities and functional mechanisms are still not fully understood (10). USPs were separated into two classes depending on their capability to bind ATP (11). Many of the USPs are expressed in response to stress, such as starvation, oxidative stress, or DNA damage (12). Genes of ATP-binding USPs are sometimes located in the same operon together with genes encoding transporters (13), and it was suggested that these USPs sense internal energy pools to regulate transporter activity (13). Here, we report on the function of the TRAP-T-associated USP TeaD. Our study reveals that TeaD regulates the internal ectoine concentration in H. elongata upon hyperosmotic stress. We have determined the crystal structure of TeaD in complex with ATP to 1.9 A˚ resolution. Furthermore, we could show that dimeric TeaD sequesters ATP during expression and that additional ATP stabilizes a dimer-dimer complex of TeaD. We discuss a regulatory mechanism of TeaD on the ectoine transport by TeaABC in H. elongata, which we extend based on genome sequence analysis to other high-affinity TRAP transporters with different substrate specificity. r 2010 American Chemical Society

Article MATERIALS AND METHODS Bacterial Strains and Growth Conditions. Cells of H. elongata DSM 2581T (DSMZ, Braunschweig, Germany) and H. elongata mutant strains KB1 (ΔectA), KB1-3 (ΔectA, ΔteaA), KB1-5 (ΔectA, ΔteaD), and AFE (ectC::Tn1732) were grown aerobically at 30 C on mineral salt medium MM63 with glucose as carbon source. For analysis by chromatography and nucleic acid isolation cells were grown in 100 mL of saline MM63 liquid medium contained in 250 mL flasks. RNA Isolation. For RNA isolation, exponentially growing cells were harvested by centrifugation, and 100 mg wet cell mass was resuspended in 4 mL of buffer (50 mM sodium acetate, 10 mM EDTA) containing 0.5 mL of SDS (10%). After addition of 5 mL of hot phenol, the sample was incubated at 65 C for 4 min and then frozen in liquid nitrogen. The frozen sample was thawed (37 C) and centrifuged (2700g) to enhance phase separation. The top layer was removed and mixed with an equal volume of phenol/chloroform/isoamyl alcohol. After centrifugation at 8000g (5 min, 4 C), the aqueous top layer was transferred to a microfuge tube, and RNA was precipitated in the cold (-70 C). The precipitated RNA was further purified using the RNeasy kit from Qiagen (Germany) according to the manufacturer’s instruction. RNA Hybridization Experiment. For the construction of a teaA-specific RNA antisense probe, an intragenic teaA DNA fragment was PCR-amplified from H. elongata DSM 2581T genomic DNA using a reverse primer (50 -ggatcctaatacgactcactatagggctcgccggtcatttcgat-30 ), which carried the promoter sequence for T7 RNA polymerase. The DNA template was applied to in vitro transcription (2 h, 37 C) to generate DIG-11UTP-labeled antisense teaA RNA. Influence of TeaD on the transcription of teaA was tested by RNA hybridization experiments using the DIG-labeled teaA antisense RNA. For that purpose, 0.5 μg of total RNA was transferred onto a nylon membrane using a vacuum slot-blot apparatus (Consort NV, Turnhout) and crosslinked by UV irradiation. Probe hybridization and chemiluminescence detection were carried out according to the DIG application manual (Roche Molecular Biochemicals, Germany). Chemiluminescence was detected by commercially available X-ray films or, in the case of a densitometric quantification, recorded with the help of a CCD camera (Fuji Luminescent Image Analyzer LAS-1000) and quantified using the software package Aida, version 2.11. To analyze the transcriptional organization of teaABCD by Northern hybridization, approximately 5 μg of total RNA was electrophoretically separated on a denaturing agarose gel (0.8%), transferred to a nylon membrane, and covalently bound to the membrane by UV irradiation. Bound RNA was hybridized with a DIG-labeled antisense teaA RNA probe for 12 h at 68 C according to the DIG application manual (Roche Molecular Biochemicals, Germany). Probe hybridization was detected by chemiluminescence via X-ray film. Promoter Mapping by 50 RACE-PCR. To identify putative promoter sequences, the transcriptional initiation sites were mapped by a modified RACE-PCR procedure using RNA from exponentially growing cells adapted to 680 mM NaCl. Transcription initiation sites of teaABCD were mapped by generating cDNA using reverse transcriptase (Superscript III; Invitrogen, USA) and reverse primer RACE-teaA1 (50 -ccgaagcgatagacctgaac-30 ). Applying terminal deoxynucleotide transferase, a poly(C) tail was attached to the 30 end, and the modified cDNA was PCR-amplified with forward primer AAP (abridged anchor

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primer; ggccacgcgtcgactagtacgggiigggiigggiig) from Invitrogen and reverse primer RACE-teaA2 (50 -aacggtatggtcggaattatc-30 ). Finally, PCR-amplified cDNA was sequenced. Determination of Cytoplasmic Ectoine by HPLC. For identification and quantification of intracellular ectoines, cells were harvested, freeze-dried, and extracted with methanol/ chloroform/water as described by Galinski and Herzog (14). Cellular extracts containing water-soluble solutes were separated by isocratic HPLC on a NH2 column using acetonitrile (80% v/v) as solvent. Eluted solutes were monitored with an UV detector at 220 nm. Purification. The gene teaD was amplified from genomic DNA that was extracted from H. elongata (15), with the primers 50 -ggatgtaaggtcatatgttcaatcggatcatgg-30 and 50 -cgggaagctttcagacgaccaggacggg-30 using the GC-rich PCR system (Roche Applied Science, Germany). The PCR product was cloned into pET22b vector (Novagen, Germany) using the NdeI and HindIII restriction sites on vector and PCR product. The resulting vector pET22b::teaD was transformed into Escherichia coli host BL21(DE3) (Novagen, Germany) for autoinduction protein expression (16). The LB medium preculture, which was grown for 6 h at 37 C, was used to inoculate autoinduction medium. The recombinant TeaD protein was synthesized for 18 h at 28 C. Cells were harvested by centrifugation at 5018g for 15 min at 4 C and resuspended in 25 mM Tris, pH 7.5, and 150 mM NaCl. The crude cell extracts were disrupted in the cell disrupter (Constant Cell Disruption) and treated with DNase (Sigma-Aldrich, Germany) and 1 mM PEFABLOC protease inhibitor (Biomol, Germany). After centrifugation (140000g, 1 h at 4 C), the supernatant was incubated in batch overnight with 10 mL of preequilibrated (25 mM Tris, pH 7.5, 150 mM NaCl) cationexchanger Sepharose (SP-Sepharose Fast Flow; GE Healthcare, USA). After washing with 25 mM Tris, pH 7.5, and 250 mM NaCl, TeaD was eluted by a gradient against 1 M NaCl and immediately dialyzed against 25 mM Tris, pH 7.5, and 250 mM NaCl to remove high salt concentration. The sample was loaded onto a Superdex 75 10/300 GL (GE Healthcare, GE Healthcare Akta-Explorer 10) in 25 mM Tris, pH 7.5, 500 mM NaCl, and optional 600 nM and 2.5 mM ATP. Buffer exchange was carried out on a PD10 column (GE Healthcare, USA) with 25 mM Tris, pH 7.5, and 150 mM MgCl2. The protein was directly used for crystallization or flash frozen in liquid nitrogen at -80 C. Determination of ADP/ATP with HPLC and ATPase Activity. To determine ATP-binding via HPLC (highperformance liquid chromatography), TeaD was purified via size exclusion chromatography against 25 mM Tris, pH 7.5, and 500 mM NaCl; afterward, the protein sample was denatured (2 min, 95 C), followed by centrifugation (1 min, 13000g). Nucleotides in the supernatant were then analyzed by HPLC using a Spherisorb ODS (octadecyl-SiO2)-1 column (Phenomenex, USA). A buffer containing 100 mM potassium phosphate, pH 6.4, 10 mM tetrabutylammonium bromide, and 7.5% acetonitrile was used as mobile phase. The detection occurred at absorption of 254 nm. The C-18 column was calibrated with a nucleotide ATP-ADP standard. ATPase activity of tetrameric TeaD was measured as described (16). Therefore, 10 μL of a 250 mM ATP solution was mixed with 10 μL of TeaD of 10 mg/mL. The reaction was started by adding 25 μL of 10% LDAO. The decrease in NADH concentration was monitored at 340 nm (Cary WIN UV; Varian, Germany) at 30 C. Blue Native Gel Electrophoresis. Purified protein was mixed with Native PAGE 4 Sample buffer (Invitrogen, USA)

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and loaded onto a 4-16% polyacrylamide gradient gel (Invitrogen, USA). A mixture of seven different proteins was used as a standard: IgM pentamer (1048 kDa), apoferritin band I (720 kDa), apoferritin band II (480 kDa), B-phycoerythrin (242 kDa), Lactate dehydrogenase (146 kDa), bovine serum albumin (66 kDa), soybean trypsin inhibitor (20 kDa) (Native Mark Unstained Protein Standard Invitrogen, USA).The anode buffer contained 50 mM Bis-Tris, pH 7, and 50 mM Tricine, 15 mM Bis-Tris, pH 7, and 0.02% Coomassie G-250 was used as cathode buffer. The blue native PAGE was performed for 30 min at 80 V and 4 h at 200 V at 4 C and fixed with a mixture of 10% acetic acid and 10% ethanol. Crystallization. Crystals of TeaD were grown at 18 C by the conventional hanging drop vapor diffusion method. The hanging drops of 300 nL size contained 4 mg/mL protein. Five millimolar ATP was added prior to crystallization. The mother liquor consisted of 100 mM Tris, pH 7.5, 200 mM (NH4)2SO4, and 15% polyethylene glycol (PEG) 3350. The crystals typically grew to dimensions of 0.4 mm  0.1 mm  0.1 mm within 7-10 days. The crystals were washed in mother liquor and flash-frozen in liquid nitrogen before exposure to X-ray. Data Collection, Structure Determination, and Refinement. All diffraction data were collected at beamline PXII at the Swiss Light Source (SLS). The data were reduced and scaled with the programs XDS and XSCALE (17). Initial phases were gained by molecular replacement with a polyglycine model of the structure of an USP of Thermus thermophilus (PDB code 2z09), available at the Protein Data Bank (http://www.rcsb.com) with the program PHASER (18) of the CCP4i suite (19); 64% of the sequences were docked in the initial map by ARP/wARP (20). Iterative rounds of manual building in COOT followed by refinement with REFMAC5 (21) including NCS and TLS were used to improve the initial model. NCS (noncrystallographic symmetry) was defined for the four chains in the asymmetric unit excluding the two flexible regions of the protein (residues 40-64 and 131-147). For TLS (translation screw libration) refinement each chain was divided into four groups, which were determined by TLSMD (21, 22). All molecular drawings were produced with PYMOL (23). Sequence and Structure Alignments. A search for similar structures of TeaD was done with DALI (24). Structural alignments were performed with TCOFFEE (25). Sequences of similar USPs were found with BLAST (26) and aligned with ClustalW (27). Sequence alignment was drawn with BioEdit (28) and ESPript (29). RESULTS AND DISCUSSION teaD Is Cotranscribed along with teaABC. To analyze the transcriptional organization of the teaABCD locus (Figure 1A), total RNA from H. elongata cells grown in minimal medium containing 690 mM NaCl was isolated and applied to Northern hybridization and RT-PCR experiments. Gene teaA, which encodes the substrate binding protein (SBP), can be transcribed separately from teaABCD, being the major transcript of the teaABCD cluster as shown by Northern hybridization (Figure 1B). Furthermore, the analyses revealed that teaABC is transcribed together with the orf teaD located downstream of teaC (Figure 1C). A similar transcription pattern was found for the TRAP transporter genes dctPQM from Rhodobacter capsulatus, where the SBP encoding dctP is also the abundant transcript when compared to the dctPQM mRNA (11). The increased mRNA level for the SBP is caused by the location of teaA and dctP, respectively, at the 50 -end of their corresponding gene

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FIGURE 1: (A) Genetic and physical organization of the teaABCD

locus. The transcription initiation sites were mapped by 50 RACEPCR, and inspection of the DNA sequences upstream of the initiation sites revealed the presence of a σ70-dependent promoter (P1) and a σS-dependent promoter (P2). (B) Total RNA from H. elongata was separated by agarose gel electrophoresis (B II) and transferred onto a nylon membrane (B I). Northern blot analysis of RNA from H. elongata wild type (WT), ΔteaC mutant KB2 (2), and the teaA deletion mutant KB1-3 (1.3) was carried out using a teaA-specific RNA probe (B I). A single transcript of approximately 1.2 kb corresponding to the calculated size of a teaA transcript was detected by hybridization with the WT RNA, while no hybridization signal was detectable with the RNA of control strain KB1-3 (ΔteaA). (C) RT-PCR analysis of teaABCD proving that teaD is cotranscribed along with teaABC. A 2400 bp PCR product was amplified from cDNA and separated by agarose gel electrophoresis (lane 8), which matched the size of the calculated teaABCD PCR product (2452 bp). cDNA synthesized from orf was only detectable by PCR with forward and reverse primers binding inside orf (lane 1) but not if the corresponding forward primer (white right-pointing arrowhead in Figure 1A) was binding to teaD (lane 4), proving that teaABCD transcription is terminated behind teaD. The same primers (lane 4) led to a teaD-orf PCR product only if chromosomal DNA was used as template (lane 3, positive control). No DNA was amplified with the primers used in lanes 1, 4, and 8 from purified total RNA prior to cDNA synthesis (lanes 2, 5, and 7, negative control), proving that the teaABCD (8) and orf (1) products were indeed amplified from cDNA and not from contaminating chromosomal DNA. Key: S, stem-loop; P1, P2, promoter; left-pointing arrowheads, forward primers for PCR; right-pointing arrowheads, reverse primers for cDNA synthesis and PCR (white arrowheads stand for no PCR product and black arrowheads for DNA amplified); > 3 3 3 52%) while they are not conserved in those TRAP transporters missing the USP gene. This suggests that these 12 proteins are specific ectoine-binding proteins, and the 12 corresponding TRAP systems probably have similar physiological properties to TeaABC. The conservation of TeaD in these systems indicates that these USPs play an important role together with the TRAP transporter in balancing the internal ectoine pool. TeaABC not only is required to accumulate external ectoine in response to osmotic stress but also functions as a salvage system for ectoine leaking out of the cell. This was found by analyzing strains of H. elongata, which possess an inoperable TeaABC transporter. These strains constantly excreted ectoine to the surrounding medium. Since uptake of compatible solutes from the medium via osmoregulated transporters will result in a decreased level of compatible solutes synthesized by the cell, it was suggested that TeaABC might be integrated (directly or indirectly) in the regulation of the cell’s compatible solute synthesis (6). Ectoine release via mechanosensitive export channels and subsequent ectoine uptake via TeaABC could serve as a signal for regulating ectoine synthesis and degradation, respectively. Since TeaD is an ATP-binding protein, it is tempting to speculate whether TeaD is modulating the osmoregulatory uptake of ectoine according to the ATP status of the cell and thereby controlling the internal

ectoine pool. However, the occurrence of TeaABCD-like TRAP systems is not always linked to the presence of an ectoine synthesis pathway. In fact, organisms such as Silicibacter pomeroyi are equipped with teaABCD but are missing the ectABC genes required for ectoine synthesis and do not use ectoine as a compatible solute. Instead, they carry the eut genes, which are thought to be involved in ectoine degradation (36), and can use ectoine as a nutrient. Besides the TeaABC transporter, two more TRAP-Ts in H. elongata are linked to USPs. One is a TAXI TRAP-T (13) whose SBP shows a high degree of sequence identity to the glutamate/ glutamine-binding protein from T. thermophilus, which structure was solved to 1 A˚ resolution (1US5). Similar glutamate transporters linked to USPs were found in several halophilic organisms, and each organism possesses a glutamate synthesis pathway. Glutamate plays an important role in osmotic stress response in H. elongata, because it is a counterion for Kþ ions, which are accumulated as osmolytes as a first response to hyperosmotic stress. In the moderately halophilic, chloride-dependent bacterium Halobacillus halophilus glutamate and glutamine are used as main compatible solutes at external salinities of 1.0-1.5 M NaCl, and biosynthesis of these solutes is regulated by chloride (37). Ectoine and glutamate are not the only osmoprotectants which are transported by a TRAP transporter linked to an USP. In Roseobacter species TRAP transporters associated to USPs were found which are specific for taurine (7), a compatible solute widespread among marine invertebrates. We assume that these three USPs are involved in the regulation of ectoine, glutamate, and taurine transport via TRAP transporters. All three show sequence conservation of several residues (magenta rhombi, Figure 9), but they differ significantly in their tetramerization region, which is located in R2, η1, and R3. It can be assumed that this region might be specifically involved in the regulatory interaction of the USP with the transporter itself or an additional interaction partner, and consequently the oligomerization interface has to be characteristic for the respective compatible solute

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FIGURE 9: Pairwise sequence alignment of USPs associated to TRAP transporters involved in ectoine (TeaD), glutamate (Gln,) and taurine (TauN) transport. Magenta rhombi label conserved residues in all USPs.

transporter system. The regulatory mechanism could involve an association/dissociation of the dimer/ATP-bound tetramer. In the dimeric state, the highly protein-interactive regions in R2, R3, and β3 would be available for a specific regulatory interaction of the USP with the transporter or, e.g., with a protein of the respective synthesis/degradation pathway, while they would be unavailable for such an interaction in a tetrameric state. Up to now it can only be speculated that ATP binding switches these TRAP-T associated USPs in an interactive state, suggesting that the internal energy pool plays a role in transporter activation. ACKNOWLEDGMENT We thank Anke Terwisscha van Scheltinga (University of € Groningen) and Ozkan Yildiz (Frankfurt, MPI) for data collection and processing assistance, Karen Davies (Frankfurt, MPI) for important discussions, Helga Volk for help with the figures, and Stefan K€oster (Frankfurt, MPI) for helpful discussions and support in purification and crystallization. We are grateful to the beamline staff at SLS PXII (Villigen, Switzerland) for excellent facilities and assistance. REFERENCES 1. Goller, K., Ofer, A., and Galinski, E. A. (1998) Construction and characterization of an NaCl-sensitive mutant of Halomonas elongata impaired in ectoine biosynthesis. FEMS Microbiol. Lett. 161, 293–300. 2. Brown, A. D. (1976) Microbial water stress. Bacteriol. Rev. 40, 803– 846. 3. Galinski, E. A., and Truper, H. G. (1982) Betaine, a compatible solute in the extremely halophilic phototropic bacterium Ectothiorhodospira halochloris. FEMS Microbiol. Lett. 13, 357–360. 4. Mackay, M. A., Norton, R. S., and Borowitzka, L. J. (1984) Organic osmoregulatory solutes in Cyanobacteria. J. Gen. Microbiol. 130, 2177–2191. 5. Wohlfarth, A., Severin, J., and Galinski, E. A. (1990) The spectrum of compatible solutes in heterotrophic halophilic Eubacteria of the family Halomonadaceae. J. Gen. Microbiol. 136, 705–712. 6. Grammann, K., Volke, A., and Kunte, H. J. (2002) New type of osmoregulated solute transporter identified in halophilic members of the bacteria domain: TRAP transporter TeaABC mediates uptake of ectoine and hydroxyectoine in Halomonas elongata DSM 2581(T). J. Bacteriol. 184, 3078–3085.

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